JP2013509166A - Process for producing polyamines - Google Patents

Abstract

The present invention relates to a method for producing a polyamine involving the use of an enzyme, in particular a method carried out in an aqueous environment, polyamines produced by the method, as well as paper production, enzyme immobilization or pharmaceutical or cosmetic composition. It relates to the use of the polyamines in the manufacture of products. The present invention also relates to a novel in-situ regeneration method for cofactor NAD (P) ⁺ .
[Selection figure] None

Description

The present invention relates to a method for producing a polyamine involving the use of an enzyme, in particular a method carried out in an aqueous environment, polyamines produced by the method, as well as paper production, enzyme immobilization or pharmaceutical or cosmetic composition. It relates to the use of the polyamines in the manufacture of products. The present invention also relates to a novel in situ regeneration method for cofactor NAD (P) ⁺ .

  Polyamines such as PEI (polyethyleneimine) are cationic polymers with a great variety of uses. PEI and its derivatives are used, for example, in the manufacture of pulp and paper to overcome the manufacturing problems caused by resins and mechanical pulp extracts (Moench D, Stange A, Linhart F, Advanced Product Concepts for Concomitant Elimination and Increased Efficiency of Paper Manufacture, Wochenblatt fuer Papierfabrikation 1996, 124: 889-892, 894-895). In addition, the polymer is charged, making it useful, for example, in immobilizing enzymes (Grunwald P, Freder R, Gunsser W, Application of polyethylene imine as carrier material for enzyme immobilization Naturwissenschaften 1981, 68: 525- 526); (de Lathouder KM, van Benthem DTJ, Wallin SA, Mateo C., Fernandez Lafuente R, Guisan JM, Kapteijn F, Moulijn JA, Polyethyleneimine (PEI) functionalized ceramic monoliths as enzyme carriers: Preparation and performance Journal of Molecular Catalysis B: Enzymatic 2008, 50: 20-27).

  Recently, progress has been made in gene transfer, and in some cases PEI combined with PEG has been shown to increase DNA / RNA cell penetration (Lungwitz U, Breunig M, Blunk T, Goepferich A, Polyethylenimine- based non-viral gene delivery systems European Journal of Pharmaceutics and Biopharmaceutics 2005, 60: 247-266); (Belenkov AI, Alakhov VY, Kabanov AV, Vinogradov SV, Panasci LC, Monia BP, Chow TY. Polyethyleneimine grafted with pluronic P85 enhances Ku86 antisense delivery and the ionizing radiation treatment efficacy in vivo. Gene Therapy 2004, 11: 1665-1672.); (Jeong JH, Kim SW, Park TG.A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles. Journal of Controled Release. 2003, 93: 183-191.). This is a very promising application since the idea of ADN (antisense oligonucleotides) was introduced and won the Nobel Prize in 2006. Successful delivery of the anticodon gene will cure many diseases such as HIV, cancer, and autoimmune responses. Still other therapeutic uses of polyamines include alleviation of symptoms associated with skin diseases and chemotherapy.

Moench D, Stange A, Linhart F, Advanced Product Concepts for Concomitant Elimination and Increased Efficiency of Paper Manufacture, Wochenblatt fuer Papierfabrikation 1996, 124: 889-892, 894-895. Grunwald P, Freder R, Gunsser W, Application of polyethylene imine as carrier material for enzyme immobilization Naturwissenschaften 1981, 68: 525-526. de Lathouder KM, van Benthem DTJ, Wallin SA, Mateo C., Fernandez Lafuente R, Guisan JM, Kapteijn F, Moulijn JA, Polyethyleneimine (PEI) functionalized ceramic monoliths as enzyme carriers: Preparation and performance Journal of Molecular Catalysis B: Enzymatic 2008 , 50: 20-27. Lungwitz U, Breunig M, Blunk T, Goepferich A, Polyethylenimine-based non-viral gene delivery systems European Journal of Pharmaceutics and Biopharmaceutics 2005, 60: 247-266. Belenkov AI, Alakhov VY, Kabanov AV, Vinogradov SV, Panasci LC, Monia BP, Chow TY.Polyethyleneimine grafted with pluronic P85 enhances Ku86 antisense delivery and the ionizing radiation treatment efficacy in vivo.Gene Therapy 2004, 11: 1665-1672. Jeong JH, Kim SW, Park TG.A new antisense oligonucleotide delivery system based on self-assembled ODN-PEG hybrid conjugate micelles. Journal of Controled Release. 2003, 93: 183-191.

  Currently available polyamines and dendrimers such as PEI are synthesized in organic solvents. Since conventional polymerization of ethyleneimine is based on ring opening, the length of the repeating unit is limited to 2 carbons between nitrogen atoms.

Therefore, the problem to be solved by the present invention is to provide a method for producing polyamines which can be carried out in an aqueous environment and is not limited to C ₂ constituents.

  The above problems have been solved by providing a novel method for the synthesis of polyamines that can be carried out in aqueous media and is based on the use of specific enzymes as further defined in the claims.

In particular, a method for synthesizing the novel polyamines schematically depicted in FIGS. 1 and 2 is provided. By the action of alcohol dehydrogenases such as horse liver alcohol dehydrogenase (HLADH) using alcohol oxidase (Figure 2) using NAD ⁺ / NADH as cofactor (Figure 1) or molecular oxygen as electron acceptor, amino alcohol, For example, 3-amino-1-propanol is oxidized to an amino aldehyde in an aqueous environment. The amino aldehyde thus formed reacts spontaneously with imines at moderately acidic pH to form polyimines. The polyimine is then converted to a polyamine, for example by reduction with sodium cyanoborohydride. The repeating unit in the polymer can have 2 or more carbon atoms between the nitrogens, can have a functional group, can be a chiral compound, and the enzyme accepts the raw amino alcohol as a substrate. Limited by ability. Many alcohol dehydrogenases and alcohol oxidases are recognized in nature, all differing in substrate specificity and / or activity.

To support complete conversion of the substrate, it is advantageous to utilize a cofactor regeneration method. To date, NAD ⁺ regeneration has been problematic and some methods utilize the enzyme NADH-oxidase, which is a rather expensive substrate (Riebel BR, Gibbs PR, Wellborn WB, Bommarius AS, Cofactor Regeneration of both NAD + from NADH and NADP + from NADPH: NADH Oxidase from Lactobacillus sanfranciscensis Advanced Synthesis & Catalysis 2003, 345: 707-712).

  The present invention provides a new and simple method based on the natural hydrolysis of vinyl acetate, which can be combined with an enzymatic polymerization reaction.

  When alcohol oxidase is used, by combining the reaction with the catalytic activity of oxidase, especially catalase, the generated hydrogen peroxide is rapidly converted to water and oxygen, thereby moving the reaction equilibrium further to the aldehyde side. , Complete conversion of the substrate is supported.

Schematic representation of polyamine synthesis catalyzed by alcohol dehydrogenase (lowering the pH to pH 4.5 enables polyimine formation. The polyimine is then reduced by a cyanoborohydride compound. Cofactor recycling is It utilizes acetaldehyde formed in situ formed at the alkaline pH or after natural hydrolysis of vinyl acetate using esterase.) Schematic representation of polyamine synthesis catalyzed by alcohol oxidase (lowering the pH to pH 4.5 enables polyimine formation. The polyimine is then reduced by a cyanoborohydride compound. The enzyme uses oxygen. Catalase further oxidizes the generated hydrogen peroxide in situ.) Size exclusion chromatograms (from top, polymer from 2-phenylglycinol, reference (PEI1800) (does not work (possibly due to over)), polymer from 3-aminopropanol, polymer from D-alaninol , Polymers from L-alaninol, each polymer having a molecular weight of about 1000 Da (molecular weight standards not shown)). It is a MALDI-TOF spectrum of an oligomer formed from 2-phenylglycinol. This is the MALDI-TOF spectrum of PEI1800.

Specific embodiments of the invention
1. In a first embodiment, the present invention provides a polyamine of the general formula (4) carried out in an aqueous reaction medium, in particular:

[Where
n is an integer of at least 1;
Residues X, independently of each other, represent a linear or branched saturated or unsaturated hydrocarbylene residue which may have one or more identical or different heteroatoms selected from N, O and S;
Residues R independently of one another represent a hydrogen atom or a linear or branched saturated or unsaturated hydrocarbyl residue optionally having one or more identical or different heteroatoms selected from N, O and S;
The terminal H ₂ N— and HO—CH ₂ — groups may be condensed to form an intramolecular amino linkage. The manufacturing method of
a) Enzymatic oxidation of an amino alcohol of the following general formula (1), which can be chiral or non-chiral, and can be used as an optically pure form or a mixture of isomers, to give the following formula (2) To the corresponding aminoaldehyde

[Wherein X and R are as defined above. ];
b) A step of polymerizing the aminoaldehyde of the formula (2) to give the following polyimine (3)

[Wherein X, R and n are as defined above. ],
c) A method is provided that preferably comprises chemically reducing the polyimine of formula (3) to the corresponding polyamine of formula (4).

2. The process according to embodiment 1, wherein said amino alcohol of step a) is a compound of formula (1) wherein X = —CH ₂ —CH ₂ —.

  3. The enzyme of stage a) is dehydrogenase and oxidase, in particular alcohol dehydrogenase EC 1.1.1.1, alcohol dehydrogenase (NADP +) 1.1.1.2, allyl-alcohol dehydrogenase EC 1.1.1.54, alcohol dehydrogenase [NAD (P) +] EC 1.1.1.71, aryl-alcohol dehydrogenase EC 1.1.1.90, aryl-alcohol dehydrogenase (NADP +) EC 1.1.1.91, 3-hydroxybenzyl-alcohol dehydrogenase EC 1.1.1.97, perillyl-alcohol dehydrogenase EC 1.1.1.144, long chain alcohol dehydrogenase EC 1.1.1.192, coniferyl-alcohol dehydrogenase EC 1.1.1.194, cinnamyl-alcohol dehydrogenase EC 1.1.1.195, cyclohexanol dehydrogenase EC 1.1.1.245, 4- (hydroxymethyl) benzenesulfonate dehydrogenase EC 1.1.1.257, 3-methyl Tanal reductase EC 1.1.1.265, S- (hydroxymethyl) glutathione dehydrogenase EC 1.1.1.284, alcohol dehydrogenase (receptor) EC 1.1.99.8, polyvinyl-alcohol dehydrogenase (receptor) EC 1.1.99.23, formaldehyde dehydrogenase (glutathione) EC1.2.1.1 and alcohol oxidase, EC1.1.3.13, aryl-alcohol oxidase EC1.1.3.7, secondary alcohol oxidase EC1.1.3.18, long-chain alcohol oxidase EC1.1.3.20, polyvinyl-alcohol oxidase EC1.1.3.30, vanillyl The method according to embodiment 1 or 2, selected from alcohol oxidase EC1.1.3.38.

  4. The enzyme is horse liver alcohol dehydrogenase (HLADH) and has the polypeptide sequence of SEQ ID NO: 1, the alcohol oxidase having the polypeptide sequence of SEQ ID NO: 2, or the sequence of SEQ ID NO: 1 or 2 The method of embodiment 3, wherein the enzyme is a mutant or variant of those enzymes having at least 60% sequence identity.

5. The method according to embodiment 3 or 4, wherein in step a), when alcohol dehydrogenase is used, NAD ⁺ is used as a cofactor for oxidation, or when alcohol oxidase is used, the reaction is aerobic.

6. The method of embodiment 5, wherein the NAD ⁺ cofactor is enzymatically regenerated by reducing the aldehyde or ketone to the corresponding alcohol with the same dehydrogenase enzyme.

  7. The method of embodiment 5, wherein the aldehyde or ketone is formed by hydrolysis of an activated ester.

8. The method according to any one of the previous embodiments, wherein the chemical reduction step c) is performed in the presence of NaBH ₃ CN or Pd / H ₂ .

  9. The molecular weight range Mn = 100 to 7,000,000, 200 to 1,000,000, 250 to 500,000, 300 to 100,000, 350 to 50,000, 400 to 10,000 or 450 to 5,000. the method of. Specific Mn ranges are 500 to 4,000 or 500 to 3,000 or 500 to 2,000.

  10. A process according to any one of the preceding embodiments, wherein step b) is preferably carried out in an aqueous medium having a pH allowing polyimine formation, in particular in the range of 3.5 to 6 or 4 to 5.

  11. The process according to any one of the previous embodiments, wherein step a) is carried out in an aqueous medium having a pH in the range 6 to 10, for example 7 to 9 or 7.5 to 8.

  12. The method according to any one of the preceding embodiments, wherein the enzyme is used in dissolved, dispersed or immobilized form.

  13. A polyamine obtained by the method according to any one of the preceding embodiments.

  14. Use of an enzyme as defined in any one of embodiments 3 and 4 in the production of a polyamine.

  15. Use of a polyamine obtained by the process of any one of embodiments 1 to 12 in the manufacture of paper, the immobilization of enzymes, or the manufacture of a pharmaceutical or cosmetic composition.

  16. Composition for improving cell penetration of DNA and / or RNA by said pharmaceutical composition; ointment for treating skin diseases; composition for treating side effects of chemotherapy; drug delivery composition, particularly topical Use of embodiment 15 selected from release agents.

17. A method for regenerating NAD (P) ⁺ produced during the formation of a first oxidation product P1 by dehydrogenase-catalyzed oxidation of a first substrate S1 of the enzyme, wherein the dehydrogenase-catalyzed oxidation proceeds in a second simultaneous manner Regenerating NAD (P) ^{+ in} combination with the formation of a second reduction product P2 different from P1 and S1 by dehydrogenase-catalyzed reduction of the second substrate S2 of the enzyme different from S1 and P1 And the substrate S2 is produced from the precursor molecule PrS2 in situ, and the precursor molecule PrS2 is decomposed naturally or through the action of a second enzyme different from the dehydrogenase.

  18. The method of embodiment 17, wherein the dehydrogenase is alcohol dehydrogenase (ADase).

  19. The method of embodiment 17 or 18, wherein said PrS2 molecule is a vinyl ester of a carboxylic acid that decomposes upon chemical or enzymatic hydrolysis to the corresponding carboxylic acid and acetaldehyde as the second substrate S2.

  20. The method of any one of embodiments 17-19, wherein the first substrate S1 is an alcohol oxidizable by the ADase.

  21. The method of embodiment 20, wherein the alcohol is an amino alcohol of formula (1) identified above.

  22. The method of any one of embodiments 18-21, wherein the regeneration reaction is performed in an aqueous medium.

Further embodiments of the invention
1. Unless otherwise specified, the following definitions apply.

  In the compounds of the above formulas (1) to (4), it is as follows.

  The parameter n is at least 1, for example 1 to 10,000, 1 to 5,000, 2 to 1,000, 3 to 800, 4 to 600, 5 to 500, 10 to 200, 15 to 100, 20 to 80 or 30 to 60, in particular It is an integer from 2 to 100, 3 to 50, 4 to 30, and 5 to 20.

Residues X may be the same or different and may have one or more, for example 1, 2 or 3 heteroatoms selected from N, O and S in the chain, linear or branched saturated or Represents an unsaturated, optionally substituted hydrocarbylene residue; the specific residue X may have 1 or 2 heteroatoms, in particular 1 O or S atom C ₁ -C ₁₀ or C ₁ -C _{5 of the} chain, such as C ₁ , C ₂ , C ₃ , C ₄ or C ₅ hydrocarbylene residues or branched C ₃ -C ₁₀ or C ₃ -C ₆ , such as An ether group or a thioether group is formed by being a C ₃ , C ₄ , C ₅ or C ₆ hydrocarbylene residue.

Residues R may be the same or different and may have one or more heteroatoms selected from N, O and S, linear or branched, saturated or unsaturated, optionally substituted hydrocarbyl residues, for example, C ₁ -C ₁₀ or C ₁ -C ₅ straight-chain, for example C _1, C _2, C _3, C ₄ or C hydrocarbyl residue or branched C _{5 3} -C ₁₀ or C ₃ - C ₆ , eg a C ₃ , C ₄ , C ₅ or C ₆ hydrocarbyl residue.

  The optional substituents for X and R are selected from hydroxy, mercapto, amino, and halogen such as F, Cl, Br.

Linear C ₁ -C ₁₀ hydrocarbyl residues include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl. Branched C ₃ -C ₁₀ hydrocarbyl residue, for example, isopropyl, isobutyl, isopentyl, 2,2-dimethylpropyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, isoheptyl, 3- Methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 2,2,3-trimethylbutyl, isooctyl, 3-methylheptyl, 4-methylheptyl, 2,2-dimethyl Hexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2,3-trimethylpentyl, 2,2,4-trimethylpentyl, 2,2,5-trimethylpentyl and isononyl.

Linear C ₁ -C ₁₀ hydrocarbylene residues include methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene and decylene. Branched C ₃ -C ₁₀ hydrocarbylene residues example, isopropylene, isobutylene, isopentylene, 2,2-dimethylpropylene, isohexylene, 3-methyl pentylene, 2,2-dimethyl butylene, 2,3-dimethyl butylene, Isoheptylene, 3-methylhexylene, 2,2-dimethylpentylene, 2,3-dimethylpentylene, 2,4-dimethylpentylene, 2,2,3-trimethylbutylene, isooctyl, 3-methylheptylene, 4-methylheptylene 2,2-dimethylhexylene, 2,4-dimethylhexylene, 2,5-dimethylhexylene, 2,2,3-trimethylpentylene, 2,2,4-trimethylpentylene, 2,2,5 -Trimethylpentylene and isononylene.

Unless otherwise noted, the expression “aminoalkanol” or “aminoaldehyde” or “polyamine” or “polyimine” used in accordance with the present invention is intended to refer to charged or uncharged and charged and uncharged forms such as ammonium salts. Refers to such compounds in a mixture. Ammonium salts can be formed with conventional counterions, such as hydroxide or halide anions, such as F ⁻ , Cl ⁻ , Br ⁻ .

  An “intermediate product” is understood as a product that is not necessarily directly detectable analytically, but is formed temporarily or continuously during a chemical or biochemical process. The “intermediate product” is removed or isolated from the process and further converted directly by a second chemical or biochemical reaction.

  As used herein, a “substantially pure” protein or enzyme is a single band after the desired purified protein is essentially polyacrylamide-sodium dodecyl sulfate gel electrophoresis (SDS-PAGE). This means that it does not contain any communicable cell components. The term “substantially pure” further describes molecules that are homogeneous due to one or more purity or homogeneity characteristics used by those skilled in the art. For example, a substantially pure protein or enzyme may have molecular weight, chromatographic transfer, amino acid composition, amino acid sequence, blocked or unblocked N-terminus, HPLC elution profile, bioactivity and other such parameters The constant and reproducible features within standard experimental deviations for parameters such as However, the term does not exclude artificial or synthetic mixtures of the protein or enzyme with other compounds. Furthermore, the term does not exclude fusion proteins comprising proteins or enzymes that may be isolated from said recombinant host.

  “Condensed” or “condensation” refers to the intermolecular or especially intramolecular formation of chemical bonds and the simultaneous formation of small molecular chemical by-products, particularly water.

  The “enzyme” reaction can be in crude form, ie, in an unpurified or nearly purified form, or in the presence of a complete microbial organism that expresses the enzyme and may place the enzyme in the extracellular space. A reaction performed under the catalytic action of at least one enzyme.

  An “aqueous reaction medium” is a liquid medium, and the liquid components are substantially a mixture of water and at least one organic liquid that can be mixed at least partially or in particular completely with water (low molecular weight alkanols such as Methanol or ethanol, or ethers such as THF, or others such as DMSO, dioxane, acetonitrile). In particular, the proportion of organic constituents can be 80% by volume or less, in particular 50, 40, 30, 20, 10 or 5% by volume or less, based on the total volume of the mixture. In particular, the amount and type of organic constituents are such that the reaction according to the invention is not adversely affected and not particularly inhibited.

  The aqueous reaction medium may further include other components, such as a buffer component, to further accelerate the reaction performed according to the present invention.

  “Regeneration” of a cofactor refers to the partial or complete reconstitution of the active form of the cofactor necessary to carry out a particular enzymatic reaction that would otherwise consume the active cofactor. The regeneration can be performed simultaneously with the enzyme reaction. That is, it can be combined with the enzyme reaction or can be performed stepwise.

  “Hydrolysis” of the ester can be catalyzed by an enzyme such as an esterase enzyme or by adjusting the pH value to a suitable pH value, for example to a pH value in the basic pH range higher than pH 7.

2. Proteins used in accordance with the present invention The present invention is not limited to the specifically mentioned proteins, but extends to their functional equivalents.

  A “functional equivalent” or “analog” or “functional mutation” of a specifically disclosed enzyme further has the desired physiological function or activity, eg, enzymatic activity, within the scope of the present invention. Its various polypeptides.

  For example, a “functional equivalent” is at least 1 to 10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% as defined herein in a test used for enzyme activity. It means an enzyme having the above or the following enzyme activity.

A “functional equivalent” according to the present invention has an amino acid different from that specifically mentioned, particularly at least one sequence position of the amino acid sequence described above, but one of the physiological activities described above. It also means a mutant having Thus, a “functional equivalent” includes mutants that can be obtained by one or more amino acid additions, substitutions, deletions and / or inversions, and the changes described can occur at any sequence position, However, it produces mutants with a profile of properties according to the invention. Functional equivalents are also provided especially when the reactive pattern occurs qualitatively simultaneously between the mutant and the unchanged polypeptide, i.e., for example, when the same substrate is converted at different rates. Examples of suitable amino acid substitutions are shown in the table below.

  “Functional equivalents” in the above sense are also “precursors” of the described polypeptides, as well as “functional derivatives” and “salts” of the polypeptides.

  A “precursor” is then a natural or synthetic precursor of a polypeptide with or without the desired physiological activity.

  The expression “salt” means a salt of a carboxyl group as well as a salt of an acid addition of an amino group of a protein molecule according to the invention. Salts of carboxyl groups can be produced by known methods, for example, inorganic salts such as sodium, calcium, ammonium, iron and zinc salts and organic bases such as amines such as triethanolamine, arginine, lysine and piperidine. There are salt and so on. Acid addition salts, such as salts with inorganic acids such as hydrochloric acid or sulfuric acid, and salts with organic acids such as acetic acid and oxalic acid are also covered by the present invention.

  A “functional derivative” of a polypeptide according to the invention can also be produced at a functional amino acid side group or at its N-terminus or C-terminus using known techniques. Such derivatives include, for example, aliphatic esters of carboxylic acid groups, ammonia or amides of carboxylic acid groups obtainable by reaction with primary or secondary amines; free amino acids produced by reaction with acyl groups N-acyl derivatives of groups; or O-acyl derivatives of free hydroxy groups produced by reaction with acyl groups.

  “Functional equivalents” naturally include polypeptides that can be obtained from other life as well as natural variants. For example, compartments of homologous sequence regions can be determined by sequence comparison, and equivalent enzymes can be determined based on specific parameters of the present invention.

  “Functional equivalents” also include, for example, fragments of the polypeptides according to the present invention that exhibit the desired physiological function, preferably individual regions or sequence motifs.

  A “functional equivalent” further includes a fusion protein having one of the above polypeptide sequences or a functional equivalent derived therefrom and at least one additional functional in functional N-terminal or C-terminal association. Different heterologous sequences (ie, without substantial mutual dysfunction of the fusion protein portion). Examples of these heterologous sequences include, but are not limited to, signal peptides, histidine anchors or enzymes.

  “Functional equivalents” further included in accordance with the present invention are homologues of the specifically disclosed proteins. These have the above percent identity values. Its value refers to the identity with the specifically disclosed amino acid sequence and should be calculated according to the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85 (8), 1988, 2444-2448. Can do. The% identity value can also be calculated from the BLAST alignment, algorithm blastp (protein-protein BLAST), or by applying the Clustal setting provided below.

  For example, identity can be calculated with the Vector NTI7.1 program suite from Informax (USA) using the clustered method with the following settings (Higgins DG, Sharp PM. Fast and Comput Appl. Biosci. 1989 Apr; 5 (2): 151-1).

Multiple alignment parameter gap start penalty: 10
Gap extension penalty: 10
Gap separation penalty range: 8
Gap Separation Penalty: Off Alignment Delay Identity%: 40
Residue specific gap: off Hydrophilic residue gap: off Transition weigh: 0.

Pairwise alignment parameters
FAST algorithm: ON
k-tuple size: 1
Gap penalty: 3
Window size: 5
Best Diagonal Number: 5.

  Alternatively, the identity is determined by Chenna, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo, Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple sequence alignment with the Clustal series of programs. (2003) Nucleic Acids Res 31 (13): 3497-500, web page: http://www.ebi.ac.uk/Tools/clustalw/index.html# and can be determined according to the following settings.

DNA gap opening penalty: 15.0
DNA gap extension penalty: 6.66
DNA matrix: identity Protein gap opening penalty: 10.0
Protein gap extension penalty: 0.2
Protein matrix: Gonnet
Protein / DNA ENDGAP: -1
Protein / DNA GAPDIST: 4.

  Percent identity of homologous polypeptides according to the present invention means in particular the percent identity of amino acid residues compared to the full length of one of the amino acid sequences specifically described herein.

  In the case of possible protein glycosylation, a “functional equivalent” according to the invention refers to a protein of the kind indicated above in a deglycosylated or glycosylated form as well as a modified form obtainable by altering the glycosylation pattern. Including.

  Such functional equivalents or homologues of a protein or polypeptide according to the invention can be produced by mutagenesis, for example by point mutation, extension or shortening of the protein.

  Such functional equivalents or homologues of the proteins according to the invention can be confirmed by screening of the combinatorial database of mutants, for example by shortening the mutants. For example, a diverse database of protein variants can be obtained by combinatorial mutation at the nucleic acid level, for example by enzymatic ligation of a mixture of synthetic oligonucleotides. There are numerous methods that can be used to obtain a database of possible homologues from degenerate oligonucleotide sequences. Chemical synthesis of degenerate gene sequences can be performed with an automated DNA synthesizer, and then the synthetic gene can be ligated with a suitable expression vector. The use of a degenerate genome makes it possible to supply all the sequences encoding the desired combination of possible protein sequences in the mixture. Methods for the synthesis of degenerate oligonucleotides are known to those skilled in the art (e.g. Narang, SA (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acids Res. 11: 477).

  In the prior art, several techniques are known for screening gene products from combinatorial databases obtained by point mutations or truncations, as well as screening cDNA libraries for gene products having specific properties. These techniques can be adapted for rapid screening of gene banks obtained by combinatorial mutations of homologues according to the present invention. The most frequently used technique for screening large gene banks based on high-throughput analysis is the cloning of gene banks with replicable expression vectors, suitable cell transformation and product detection with the resulting vector database. The expression of the combinatorial gene under conditions that facilitate the detection of the desired activity to isolate the vector encoding the desired gene. Functional ensemble mutagenesis (REM), a technique that increases the frequency of functional mutants in the database, can be used in conjunction with screening tests to confirm homologs (Arkin and Yourvan (1992) PNAS 89: 7811 -7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331).

3. Further embodiments of the method of the invention
3.1. Overview The process of the present invention can be carried out in batch, semi-batch or continuous mode.

  Enzyme or enzymes that can be present during the method of the present invention are viable cells, harvested cells, dead cells, permeabilized cells, crude cell extracts, partially purified extracts that produce the enzyme or enzymes. It can be present in or in pure (about 90 to 99.5 wt%) or complete (about 99.5 to 99.9 wt%, or 100 wt%) (per total dry weight of the enzyme preparation, respectively) in pure form.

  The enzyme may be used in a free state or may be immobilized. The immobilized enzyme is immobilized on a suitable inert carrier. Suitable carriers are described, for example, in EP-A-1149849, EP-A-1069183 and DE-OS100193773 and other documents cited therein. Suitable carriers are, for example, clays (kaolinite, perlite, etc.), silicon dioxide, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchanger, synthetic polymer (polystyrene etc.), acrylic resin, phenol formaldehyde resin, Polyurethanes and polyolefins (such as polyethylenes and polypropylenes). The carrier is usually in particulate form and preferentially used in porous form. The particle size is usually in the range of 5 mm or less or 2 mm or less. The enzyme or cell can also be cross-linked with glutaraldehyde. Suitable immobilization techniques are described, for example, in J. Lalonde and A. Margolin “Immobilization of Enzymes”, K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Have been described.

  The method according to the invention is a general reactor known to the person skilled in the art, for example varying from laboratory scale (reaction volumes of several milliliters to tens of liters) to industrial scale (reaction volumes of several liters to several thousand cubic meters). Can be done in a range of scales.

  When the enzyme is used in an immobilized form, a reactor can be used. The reactor usually allows control of the amount of at least one enzyme, the amount of at least one substrate, the pH, temperature and circulation of the reaction medium. If at least one enzyme is present in a living cell, the method will be a fermentation. In this case, biocatalyst production takes place in a bioreactor (fermentor), in which case the parameters necessary for the living conditions suitable for viable cells (e.g. nutrient media, temperature, aeration, presence of oxygen or other gases or Non-existence, antibiotics, etc.) can be controlled. The person skilled in the art is familiar with chemical reactors or bioreactors, for example with procedures for scaling up chemical or biotechnological methods from laboratory to industrial scale or optimizing process parameters. It is also described extensively in the literature (for biotechnology methods see, for example, Crueger und Crueger, Biotechnologie-Lehrbuch der angewandten Mikrobiologie, 2. Aufl., R. Oldenbourg Verlag, Muenchen, Wien, 1984; Or see Biotechnology, Volume 3, 2nd Edition, Rehm et al. Eds., (1993), especially Chapter II.)

  Cells containing at least one enzyme of the present invention may be obtained by physical or mechanical means such as ultrasonic or radio frequency pulses, French press, or chemical means such as hypotonic media, lytic enzymes and detergents present in the medium, Alternatively, the permeabilization treatment can be performed by a combination of such methods. Examples of surfactants include digitonin, n-dodecyl maltoside, octyl glycoside, Triton® X-100, Tween® 20, deoxycholate, CHAPS (3-[(3-colamide Propyl) dimethylammonio] -1-propanesulfonate), Nonidet® P40 (ethylphenol poly (ethylene glycol ether)), and the like.

  A suitable enzyme content can be readily determined by one skilled in the art by performing a limited number of optimization experiments.

3.2 pH value and buffer
3.2.1 Oxidation stage a)
In the process of the present invention, an enantiomerically pure or chiral substrate in pure form or a stereoisomer mixture exhibiting one stereoisomer enrichment can be used.

  Conversion of a substrate according to general formula (1) to an aminoaldehyde according to general formula (2) usually depends on the conditions optimal for the enzyme (dehydrogenase or oxidase) or enzyme system (in combination with a cofactor that regenerates the enzyme). At a pH of about 6.0 to about 10.0, such as a pH of about 7 to 9.5. The optimum pH value can be easily determined by conducting a limited number of general experiments in advance. For example, when using Pichia pastoris alcohol oxidase, the pH is in the range of 6.5 to 7.5, and even in the range of about 7.0. Alternatively, alcohol dehydrogenase from Equus caballus requires a more alkaline pH, and the reaction can be performed in the range of about 8.5 to 9.5, or even about 9.0.

  Suitable buffers for the above pH values or pH ranges can be used, such as MOPS, HEPES, PIPES, phosphate, borate and Tris buffers. Tris buffer can be used in the pH range of 7.5 to 9.0, and phosphate or MOPS buffer can be used in the pH range of 6.0 to 8.0. The concentration of the buffering agent can vary depending on the process conditions and can range from 1 mM to 200 mM, such as from 1 mM to 100 mM, 1 mM to 50 mM. In particular, the buffer concentration can be 2 mM to 25 mM or 3 mM to 10 mM, for example 5 mM.

3.2.2 Polyimine formation stage b)
To induce polymerization, the pH of the reaction medium is lowered by adding a suitable inorganic or organic acid. For example, an inorganic acid such as H ₂ SO ₄ or HCl can be added. The pH is lowered to reach a range that helps the polymerization of the aminoaldehyde monomer. Usually, the pH can be in the range of about 3.5 to 5.5, in particular 4 to 5. The acid can be added to the reaction mixture obtained from step a), or by adding one or more components, for example gradually or a suitable quencher such as EDTA or hydrogen peroxide, or cofactor regeneration It is also possible to carry out using the reaction mixture of step a) after removing the protein material resulting from enzyme deactivation that occurs by removing the unconverted reactants or co-reactants required for the step. Usually, however, no pretreatment is necessary to carry out step b).

3.2.3 Reduction stage c)
The reduction can be performed after step b) or simultaneously. A suitable reducing agent for reducing the polyimine to the corresponding polyamine in aqueous solution, such as sodium cyanoborohydride or sodium borohydride, sodium triacetoxyborohydride, H ₂ / PdC, the desired degree of imine groups Is added in an amount sufficient to provide (preferably complete) hydrogenation. For example, the reducing agent can be added in a 2 to 10 fold molar excess.

3.2.4 NAD (P) ⁺ cofactor regeneration Suitable pH and buffer conditions for cofactor regeneration include the conditions of step a) above when cofactor regeneration is combined with such aminoaldehyde formation step a). It can be substantially the same. When combined with oxidoreductases and other cofactors that consume different substrates, one skilled in the art can adjust the specific process conditions for a new system, which is a limited number of routine experiments It will be clear that this can be done.

3.3. Temperature and duration of the reaction The process steps of the present invention can be carried out at a temperature where the enzyme used is tolerated or assists the intended chemical or enzymatic conversion reaction used.

  The temperature of the enzymatic conversion can be correlated with the optimal growth temperature of the organism or microorganism that has the enzyme or is used as an extraction source, but can be readily determined by one skilled in the art.

  In general, the process steps can be carried out at temperatures of 20 ° C. to 70 ° C., in particular 40 ° C. to 50 ° C. Examples of reaction temperatures include about 30 ° C, about 35 ° C, about 37 ° C, about 40 ° C, about 45 ° C, about 50 ° C, about 55 ° C and about 60 ° C.

  Each step can proceed until an equilibrium between the substrate and the corresponding product is achieved, but can be stopped earlier. Typical reaction times are in the range of about 1 minute to 25 hours, especially in the range of about 10 minutes to 6 hours, or in the range of about 1 hour to 4 hours.

3.4 Cosolvent If necessary, one or more organic cosolvents can be included in the reaction medium to increase the solubility of the substrate. Examples of suitable cosolvents include butan-2-ol, methyl-tert-butyl ether (MTBE) and dimethyl sulfoxide (DMSO). For the co-solvent, a concentration below saturation in the aqueous medium can be used, for example, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, or about 1%. It is as follows.

3.5 Other reactants Additional reactants or co-reactants necessary to carry out the reactions of the present invention (substrate conversion, cofactor regeneration) are added to the reaction system as necessary and in suitable amounts. Also good.

For example, the cofactor (NAD (P) ⁺ ) can be added in non-stoichiometric amounts, for example in the range of 0.001 to 10, 0.01 to 5, or 0.1 to 1 mM.

  For example, an activated ester for cofactor regeneration such as vinyl acetate can be added in a suitable amount, for example in an amount ranging from 1 to 10 times or 2 to 5 times a molar excess compared to aminoalcohol.

  For example, hydrogen peroxide can be added in a suitable amount, for example in an amount ranging from 0.0001 to 10, 0.001 to 10, 0.01 to 5 or 0.1 to 1 mM.

3.6 Polymer Isolation The method of the present invention can further include a step of polymer recovery. The term “recovery” includes extraction, collection, isolation or purification of the compound from the reaction medium. Compound recovery can be achieved by treatment with conventional resins (for example, anion or cation exchange resin, nonionic adsorption resin, etc.), treatment with conventional adsorbent (for example, activated carbon, silicic acid, silica gel, cellulose, alumina, etc.) PH change, solvent extraction (e.g. with conventional solvents such as alcohol, ethyl acetate, hexane), distillation, dialysis, filtration, concentration, crystallization, recrystallization, lyophilization, etc. and combinations thereof (limited to these) Can be performed according to conventional isolation or purification methods known in the art.

  For example, the polymer can be recovered from the reaction solution by first removing residual microorganisms or proteins, or the solubility of the polymer in an aqueous environment if substantially no such impurities are present. Can be recovered by extraction with a suitable organic solvent, preferably a substantially water-insoluble solvent (repeat as appropriate). Suitable solvents are for example but not limited to dichloromethane, toluene, methylene chloride, butyl acetate, diisopropyl ether, benzene, MTBE or ethyl acetate.

  Extraction can be performed after increasing the pH of the mixture to about pH 12 to 14 by adding a suitable base such as NaOH and adjusting the ionic strength by adding an aqueous salt solution such as saturated saline.

  The following examples are only intended to illustrate the present invention. Many possible variations that will be apparent to those skilled in the art are also within the scope of the present invention.

Experimental part
Example 1 Production of Polyamines in Aqueous Medium with Horse Liver Alcohol Dehydrogenase (HLADH) or Alcohol Oxidase (AOX) Two different approaches for polyamine synthesis in the aqueous medium are shown schematically in FIGS. 1 and 2. I'm drawing.

In the HLADH method, cofactor regeneration is performed using vinyl acetate. Vinyl acetate hydrolyzes rapidly at pH 9.1, breaking down into acetic acid and acetaldehyde. The aldehyde can be reduced by HLADH to ethanol, thereby forming NAD ⁺ from NADH. Unreacted acetaldehyde can be removed by bubbling N ₂ after inactivating the enzyme with EDTA or H ₂ O ₂ . For the alcohol dehydrogenase HLADH, the reaction was followed spectrophotometrically by NADH production at 340 nm. Michaelis-Menten rate constants were obtained using different concentrations of various amino alcohols.

  The AOX method does not require cofactor regeneration. The reaction is further driven by catalase catalyzed decomposition of hydrogen peroxide. The AOX reaction is spectrophotometrically determined by following the formation of hydrogen peroxide by addition of peroxidase and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) at 420 nm be able to.

a) Materials The chemicals used in this experiment were of grade> 98% purity and were purchased from Sigma Aldrich.

  Since AOX (Pichia pastoris) used has no activity against aminoalcohol at alkaline pH, synthesis was performed at pH 7.0. The enzyme was obtained commercially from Sigma Aldrich.

  Alcohol dehydrogenase (Equus caballus) was not active at neutral pH and had a good reaction rate at alkaline pH. This reaction was performed at pH 9.1. The enzyme was obtained commercially from Sigma Aldrich.

b) Polyamine synthesis
b1) Polyamine synthesis with AOX
An amino alcohol selected from 3-aminopropanol, D-alaninol, L-alaninol or 2-phenylglycinol is dissolved in 100 mM MOPS-buffer to a concentration of 5 mM to 3 M with a volume of 1 mL to 300 mL and a pH of 7.0 with HCl. Set to. AOX and catalase were added and the solution was incubated at 25 ° C. for 48 hours. The amount of AOX was adjusted to the amount necessary to complete the conversion within 24 hours, resulting in a catalase in excess of at least 10-fold enzyme unit. The pH was lowered to 4.5 by adding acetic acid and 3 equivalents of NaCNBH ₃ was added.

b2) Polyamine synthesis by HLADH The amino alcohol was dissolved in water at 1 mL: 300 mL to a concentration of 5 mM to 3 M with a 2-fold molar excess of vinyl acetate, and the pH was set to 9.1 with NaOH / HCl. The substrate itself has a buffering capacity at this pH. ADH (alcohol dehydrogenase) and NAD ⁺ (0.5 mM) were added and the solution was incubated at 25 ° C. for 48 hours. The amount of enzyme was adjusted to complete the conversion within 24 hours.

Thereafter, 30% H ₂ O ₂ was added to a peroxide concentration of 100 equivalents with respect to the enzyme, and the reaction solution was incubated for 4 hours. Acetaldehyde was removed by blowing N ₂ for 4 hours with a syringe immersed in the mixture. The pH was lowered to 4.5 by adding HCl and 3 equivalents of NaCNBH ₃ was added.

c) Product extraction The reaction mixture was mixed with 1 equivalent of saturated brine and the pH was set to 14 by adding NaOH. Thereafter, the polymer / oligomer was separated from the aqueous solution by repeatedly shaking with dichloromethane. The dichloromethane phase was then lyophilized to give a viscous product, which is an oligo / polyamine.

d) The characterization product (produced polymer / oligomer) was dissolved in dimethyl sulfoxide and subjected to size exclusion chromatography (SEC).

  Alternatively, various amounts of the acquisition (manufactured polymer / oligomer) ranging from 1 μL to 200 μL were prepared in acetonitrile / water (1: 1 with 3% trifluoroacetic acid and 10 g / L 2,5-dihydroxybenzoic acid. ) Mass analysis by matrix-assisted laser desorption ionization (MALDI) was performed by dissolving in 0.5 mL of solution and measuring ion mass by time of flight (TOF).

e) Results and Discussion The molecular weight of the polyamine compound is determined by the degree of aminoaldehyde formation. In the case of the reaction using HLADH, an excess amount of vinyl acetate was added to completely convert the substrate, and the acetaldehyde formed was removed by blowing N ₂ into the mixture. When AOX is used, complete conversion is ensured by the dissolution of oxygen from the ambient air and the action of catalase.

  To form an imine, the pH is lowered to 4.5 by adding acetic acid and / or HCl. When alcohol dehydrogenase is used, HCl can be used in this case because acetic acid produced from hydrolysis of the activated ester functions as a buffer at this pH.

The results of the conversion of various amino alcohols catalyzed by AOX are summarized in Table 1, and the rate constants are also shown in the table.

  The reaction was followed spectrophotometrically at 420 nm via the formation of hydrogen peroxide by adding peroxidase and ABTS.

E value = proportionality relative to the other of one enantiomer as a substrate defined by the division of the specificity constant. The specificity constant is defined as k _cat / K _M.

  In the case of 2-amino-1-phenylethanol (2-phenylglycinol), the assay could not be used because the substrate precipitated upon addition of ABTS. Other experiments in which ABTS was replaced with pyrogallol or pullpald were used to measure aldehyde formation but were not definitive. However, the experiment revealed that the enzyme was catalyzing the reaction and the appearance of an aldehyde. The rate reproducibility in these experiments was not satisfactory and kinetic data could not be extracted.

The results for the conversion of various amino alcohols catalyzed by HLADH are summarized in Table 2, which also shows rate constants.

  The reaction catalyzed by HLADH was followed spectrophotometrically at 340 nm due to the formation of NADH.

  After extraction with dichloromethane (mixed aqueous solution with 1 equivalent of saturated saline and pH set to 14) and after evaporation of the solvent, the material is subjected to size exclusion chromatography (SEC) after dissolution in DMSO I was able to. FIG. 3 shows the chromatogram.

  The polydispersity was estimated to vary between 1.04 and 1.07 (performed by the software Millenium).

  Mass spectrometry could also be performed on the extract. The material was dissolved at various concentrations in water / acetonitrile (1: 1) containing 3% TFA and 10 g / L 2,5-dihydroxybenzoic acid. The spectrum of the polymer formed from 2-phenylglycinol is shown in FIG.

  Presence of subunits having no phenyl group is presumed, and it is presumed to be derived from contaminants in the raw material. If alaninol is present as a contaminant and is a better substrate than 2-phenylglycinol, it is believed that it is incorporated before the remaining molecules are converted to aldehydes. Such a subunit corresponds to a mass difference of 57.06, and a subunit having a phenyl group corresponds to a mass difference of 119.07. These mass differences can be used to explain the difference in peak mass in the spectrum of FIG.

  A commercially available PEI1800 was also examined by MALDI-TOF, and the spectrum is shown in FIG. In FIG. 5, there are several series of polymers, resulting in a fairly unclear spectrum. The mass difference between the peaks is about 43 as expected.

Claims (22)

A polyamine of the following general formula (4):

[Where
n is an integer of at least 1;
Residues X, independently of each other, represent a linear or branched saturated or unsaturated hydrocarbylene residue which may have one or more identical or different heteroatoms selected from N, O and S;
Residues R independently of one another represent a hydrogen atom or a linear or branched saturated or unsaturated hydrocarbyl residue optionally having one or more identical or different heteroatoms selected from N, O and S;
The terminal H ₂ N— and HO—CH ₂ — groups may be condensed to form an intramolecular amino linkage. The manufacturing method of
a) Enzymatic oxidation of an amino alcohol of the following general formula (1), which can be chiral or non-chiral, and can be used as an optically pure form or a mixture of isomers, to give the following formula (2) To the corresponding aminoaldehyde

[Wherein X and R are as defined above. ];
b) A step of polymerizing the aminoaldehyde of the formula (2) to give the following polyimine (3)

[Wherein X, R and n are as defined above. ],
c) A method comprising the step of reducing the polyimine of formula (3) to the corresponding polyamine of formula (4). The process according to claim 1, wherein the amino alcohol of step a) is a compound of formula (1) wherein X = -CH ₂ -CH _2- .   The enzymes of step a) are dehydrogenases and oxidases, in particular alcohol dehydrogenase EC 1.1.1.1, alcohol dehydrogenase (NADP +) 1.1.1.2, allyl-alcohol dehydrogenase EC 1.1.1.54, alcohol dehydrogenase [NAD (P) +] EC 1.1. 1.71, aryl-alcohol dehydrogenase EC 1.1.1.90, aryl-alcohol dehydrogenase (NADP +) EC 1.1.1.91, 3-hydroxybenzyl-alcohol dehydrogenase EC 1.1.1.97, perillyl-alcohol dehydrogenase EC 1.1.1.144, long chain alcohol dehydrogenase EC 1.1 .1.192, coniferyl-alcohol dehydrogenase EC 1.1.1.194, cinnamyl-alcohol dehydrogenase EC 1.1.1.195, cyclohexanol dehydrogenase EC 1.1.1.245, 4- (hydroxymethyl) benzenesulfonate dehydrogenase EC 1.1.1.257, 3-methyl Tanal reductase EC 1.1.1.265, S- (hydroxymethyl) glutathione dehydrogenase EC 1.1.1.284, alcohol dehydrogenase (receptor) EC 1.1.99.8, polyvinyl-alcohol dehydrogenase (receptor) EC 1.1.99.23, formaldehyde dehydrogenase (glutathione) EC1.2.1.1 and alcohol oxidase, EC1.1.3.13, aryl-alcohol oxidase EC1.1.3.7, secondary alcohol oxidase EC1.1.3.18, long-chain alcohol oxidase EC1.1.3.20, polyvinyl-alcohol oxidase EC1.1.3.30, vanillyl -Method according to claim 1 or 2, selected from alcohol oxidase EC1.1.3.38.   The enzyme is horse liver alcohol dehydrogenase (HLADH) having the polypeptide sequence of SEQ ID NO: 1, the alcohol oxidase having the polypeptide sequence of SEQ ID NO: 2, or at least relative to the sequence of SEQ ID NO: 1 or 2. 4. The method according to claim 3, which is a mutant or variant of those enzymes having 60% sequence identity. The method according to claim 3 or 4, wherein in step a), when alcohol dehydrogenase is used, NAD ⁺ is used as a cofactor for oxidation, or when alcohol oxidase is used, the reaction is aerobic. 6. The method of claim 5, wherein the NAD ⁺ cofactor is enzymatically regenerated by reducing the aldehyde or ketone to the corresponding alcohol with the same dehydrogenase enzyme.   6. The method of claim 5, wherein the aldehyde or ketone is formed by hydrolysis of an activated ester. Chemical reduction step c) carried out in the presence of NaBH ₃ CN or Pd / H _2, the method according to any one of claims 1 to 7.   9. A process as claimed in claim 1, wherein polyamines having a molecular weight range Mn = 100 to 7,000,000 are obtained.   A process according to any one of the preceding claims, wherein step b) is carried out in an aqueous medium having a pH allowing polyimine formation, preferably in the range of 3.5 to 6.   The process according to any one of claims 1 to 10, wherein step a) is carried out in an aqueous medium having a pH in the range of 6 to 10.   The method according to any one of claims 1 to 11, wherein the enzyme is used in a dissolved, dispersed or immobilized form.   The polyamine obtained by the method of any one of Claims 1-12.   Use of an enzyme as defined in any one of claims 3 and 4 in the production of polyamines.   Use of a polyamine obtained by the process according to any one of claims 1 to 12 in the manufacture of paper, the immobilization of enzymes, or the manufacture of a pharmaceutical or cosmetic composition.   A composition for improving cell penetration of DNA and / or RNA; an ointment for treating skin diseases; a composition for treating side effects of chemotherapy; a drug delivery composition. Use as described in 15. A method for regenerating NAD (P) ⁺ produced during the formation of a first oxidation product P1 by a dehydrogenase-catalyzed oxidation reaction of a first substrate S1 of the enzyme, wherein the dehydrogenase-catalyzed oxidation reaction proceeds a second simultaneously S1 And regenerating NAD (P) ^{+ in} combination with the formation of a second reduction product P2 different from P1 and S1 by dehydrogenase-catalyzed reduction of the second substrate S2 of the enzyme different from P1 A regeneration method for producing the substrate S2 from the precursor molecule PrS2.   The method according to claim 17, wherein the dehydrogenase is alcohol dehydrogenase (ADase).   19. The method according to claim 17, wherein the PrS2 molecule is a vinyl ester of a carboxylic acid that decomposes upon hydrolysis to become the corresponding carboxylic acid and acetaldehyde as the second substrate S2.   20. The method according to any one of claims 17 to 19, wherein the first substrate S1 is an alcohol that can be oxidized by the ADase.   21. The method of claim 20, wherein the alcohol is an amino alcohol of formula (1) identified above.   The method according to any one of claims 18 to 21, wherein the regeneration reaction is carried out in an aqueous medium.

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